Frank Neipel

Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins.

The passage of mammalian cells through the restriction point into the S phase of the cell cycle is regulated by the activities of Cdk4 and Cdk6 complexed with the D-type cyclins and by cyclin E/Cdk2 (refs 1,2,3). The activities of these holoenzymes are constrained by CDK inhibitory proteins. The importance of the restriction point is illustrated by its deregulation in many tumour cells and upon infection with DNA tumour viruses. Here we describe the properties of cyclins encoded by two herpesviruses, herpesvirus saimiri (HVS) which can transform blood lymphocytes and induce malignancies of lymphoid origin in New World primates, and human herpesvirus 8 (HHV8) implicated as a causative agent of Kaposis sarcoma and body cavity lymphomas. Both viral cyclins form active kinase complexes with Cdk6 that are resistant to inhibition by the CDK inhibitors p16Ink4a, p21Cip1and p27Kip1. Furthermore, ectopic expression of a viral cyclin prevents G1 arrest imposed by each inhibitor and stimulates cell-cycle progression in quiescent fibroblasts. These results suggest a new mechanism for deregulation of the cell cycle and indicate that the viral cyclins may contribute to the oncogenic nature of these viruses.

Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma

Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposis sarcoma‐associated herpes virus (KSHV) or human herpes virus‐8 (HHV‐8) and the development of Kaposis sarcoma (KS). To clarify the role of HHV‐8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7‐kb HHV‐8‐encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS‐KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV‐8‐infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL‐1 established from a body cavity‐based lymphoma (BCBL) and latently infected with HHV‐8. In all KS biopsies examined, independent of their epidemiological type, the late‐stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV‐8 infection of these cells. The presence of T0.7‐expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV‐8 infection in KS pathogenesis. Int. J. Cancer 72:68–71, 1997.

Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1

ABSTRACT An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposis sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1ΔTMFc). K8.1ΔTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1ΔTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.

The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Kaposis sarcoma–associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma, a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies. A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells. We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA2 and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry. Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposis sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposis sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.

Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein.

Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposis sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS binding and DNA binding. Moreover, ORF52 homologs in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52 null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing.

The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5

Kaposis sarcoma-associated herpesvirus encodes four genes with homology to the family of interferon regulatory factors (IRFs). At least one of these viral IRFs, vIRF-3, is expressed in latently Kaposis sarcoma-associated herpesvirus-infected primary effusion lymphoma (PEL) cells and is essential for the survival of PEL cells. We now report that vIRF-3 interacts with cellular IRF-5, thereby inhibiting binding of IRF-5 to interferon-responsive promoter elements. Consequently, vIRF-3 blocked IRF-5-mediated promoter activation. A central double helix motif present in vIRF-3 was sufficient to abrogate both DNA binding and transcriptional transactivation by IRF-5. Upon DNA damage or activation of the interferon or Toll-like receptor pathways, cytoplasmic IRF-5 has been reported to be translocated to the nucleus, which results in induction of both p53-independent apoptosis and p21-mediated cell cycle arrest. We report here that IRF-5 is present in the nuclei of PEL cells without interferon stimulation. Silencing of vIRF-3 expression in PEL cells was accompanied by increased sensitivity to interferon-mediated apoptosis and up-regulation of IRF-5 target genes. In addition, vIRF-3 antagonized IRF-5-mediated activation of the p21 promoter. The data presented here indicate that vIRF-3 contributes to immune evasion and sustained proliferation of PEL cells by releasing IRF-5 from transcription complexes.

The unique region of the human herpesvirus 6 genome is essentially collinear with the UL segment of human cytomegalovirus

The entire genome of human herpesvirus 6 (HHV-6) strain U1102 was cloned as overlapping fragments in cosmid and plasmid vectors. Cleavage maps were constructed for the restriction endonucleases BamHI, EcoRI, NotI and SmaI. The genome of HHV-6 U1102 is a linear dsDNA of 163 kbp, consisting of a long unique 142 kbp region flanked by direct terminal repeats of 10.5 kbp. Short stretches (290 to 470 nucleotides) of DNA, four from the terminal repeats and 55 from the UL region, were sequenced and compared by computer with the known herpesvirus amino acid sequences. Homologies were found for 10 open reading frames that are scattered over the UL region of HHV-6. Their relative positions and orientations indicate that the unique region of HHV-6 is essentially collinear with the UL region of human cytomegalovirus (HCMV), but is not collinear with the other human herpesviruses. It confirms and extends earlier observations that HHV-6 is more closely related to the beta-herpesvirus HCMV, suggesting that HHV-6 may be considered as the prototype of a new beta 2-herpesvirus subgroup.

HIV entry inhibition by the envelope 2 glycoprotein of GB virus C.

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.

Intracellular Localization Map of Human Herpesvirus 8 Proteins

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposis sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 3 Inhibits Gamma Interferon and Major Histocompatibility Complex Class II Expression

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) carries four genes with homology to human interferon regulatory factors (IRFs). One of these IRFs, the viral interferon regulatory factor 3 (vIRF-3), is expressed in latently infected primary effusion lymphoma (PEL) cells and required for their continuous proliferation. Moreover, vIRF-3 is known to be involved in modulation of the type I interferon (IFN) response. We now show that vIRF-3 also interferes with the type II interferon system and antigen presentation to the adaptive immune system. Starting with an analysis of the transcriptome, we show that vIRF-3 inhibits expression of major histocompatibility complex class II (MHC II) molecules: small interfering RNA (siRNA)-mediated knockdown of vIRF-3 in KSHV-infected PEL cell lines resulted in increased MHC II levels; overexpression of vIRF-3 in KSHV-negative B cells leads to downmodulation of MHC II. This regulation could be traced back to inhibition of class II transactivator (CIITA) transcription by vIRF-3. Reporter assays revealed that the gamma interferon (IFN-γ)-sensitive CIITA promoters PIV and PIII were inhibited by vIRF-3. Consistently, IFN-γ levels increased upon vIRF-3 knockdown in PEL cells. IFN-γ regulation by vIRF-3 was confirmed in reporter assays as well as by upregulation of typical IFN-γ target genes upon knockdown of vIRF-3 in PEL cells. In summary, we conclude that vIRF-3 contributes to the viral immunoevasion by downregulation of IFN-γ and CIITA and thus MHC II expression.